Defeating peripheral neuropathy The mechanisms underlying peripheral neuropathies are not well understood. Spaulding et al . studied mouse models of the inherited Charcot-Marie-Tooth (CMT) disease, which is caused by mutations in transfer RNA (tRNA) synthetases. Changes in gene expression and the rate of protein synthesis in neurons in the spinal cord triggered the cell stress response activated by the protein sensor GCN2. When GCN2 was genetically deleted or inhibited with drugs, the stress response was blocked, and the neuropathy was much milder. Zuko et al . found that mutant glycyl-tRNA synthetases bind tRNA Gly but fail to release it, thus depleting the cellular tRNA Gly pool. This process caused stalling of translating ribosomes on glycine codons and activated the integrated stress response. Transgenic tRNA Gly overexpression prevented peripheral neuropathy and protein synthesis defects in mouse and fruit fly models. Thus, elevating tRNA Gly levels or targeting GCN2 may have therapeutic potential for this currently untreatable disease (see the Perspective by Mellado and Willis). —SMH

Exposure to genotoxic stress by environmental agents or treatments, such as radiation therapy, can diminish healthspan and accelerate aging. We have developed a Drosophila melanogaster model to study the molecular effects of radiation-induced damage and repair. Utilizing a quantitative intestinal permeability assay, we performed an unbiased GWAS screen (using 156 strains from the Drosophila Genetic Reference Panel) to search for natural genetic variants that regulate radiation-induced gut permeability in adult D. melanogaster. From this screen, we identified an RNA binding protein, Musashi (msi), as one of the possible genes associated with changes in intestinal permeability upon radiation. The overexpression of msi promoted intestinal stem cell proliferation, which increased survival after irradiation and rescued radiation-induced intestinal permeability. In summary, we have established D. melanogaster as an expedient model system to study the effects of radiation-induced damage to the intestine in adults and have identified msi as a potential therapeutic target.

The underlying causes of aging remain elusive, but may include decreased intestinal homeostasis followed by disruption of the intestinal barrier, which can be mimicked by nutrient‐rich diets. S3QELs are small‐molecule suppressors of site IIIQo electron leak; they suppress superoxide generation at complex III of the mitochondrial electron transport chain without inhibiting oxidative phosphorylation. Here we show that feeding different S3QELs to Drosophila on a high‐nutrient diet protects against greater intestinal permeability, greater enterocyte apoptotic cell number, and shorter median lifespan. Hif‐1α knockdown in enterocytes also protects, and blunts any further protection by S3QELs. Feeding S3QELs to mice on a high‐fat diet also protects against the diet‐induced increase in intestinal permeability. Our results demonstrate by inference of S3QEL use that superoxide produced by complex III in enterocytes contributes to diet‐induced intestinal barrier disruption in both flies and mice.

Exposure to genotoxic environmental agents has detrimental effects on health span and aging. Macromolecular damage caused by these factors is also known to lead to diseases like cancer. Radiation therapy is the treatment of choice for malignant tumors and several non-malignant diseases. Despite its effectiveness, it often leads to unintended complications. Radio-sensitivity of healthy ‘bystander’ cells is an important factor in mediating these complications, which often result in several long-term and debilitating side effects. This incurs huge costs in patient care to maintain the well-being of survivors. Current efforts to understand the underlying molecular patho-physiology of radiation damage involve the use of in vitro human cell or rodent models. However, these methods have limitations, as in vitro models cannot perfectly mimic complex tissue micro-environments and in vivo rodent models are far too expensive, time-consuming, and restricted. To fill this critical gap, Drosophila melanogaster has been used as it is a genetically malleable and an inexpensive model to study radiation-induced damage. The conservation between humans and flies at the cellular and molecular levels allowed researchers to study similarities between flies and mammals in how they respond to radiation. DNA damage resulting from radiation exposure inhibits not only intestinal stem cell (ISC) proliferation but also causes extensive apoptosis in the enterocytes leading to increased intestinal permeability and reduced survival. To identify novel regulatory pathways that can alleviate radiation induced intestinal damage, a screen was performed using approximately strains from the Drosophila Genetic Reference Panel (DGRP). This Genome Wide Association Study (GWAS) analysis identified several candidate genes. In this thesis, two of the top candidate genes and their role in regulating intestinal damage caused by radiation were characterized. In the first aim, Meltrin was characterized for the effect of its tissue-specific knockdown in the clearance of damaged Enterocytes (EC), the main absorptive cells in fly intestine. This characterization was achieved by measuring survival, gut damage, and prevalence of apoptotic cells in irradiated guts. In the second aim, the Msi was investigated for its role in reparative proliferation of intestinal stem cells (ISCs). Results from this thesis project have identified two new conserved genes involved in radiation damage repair with potential for future relevance as therapeutic targets.

BackgroundCharcot‐Marie‐Tooth disease type 1X is caused by mutations in GJB1, which is the second most common gene associated with inherited peripheral neuropathy. The GJB1 gene encodes connexin 32 (CX32), a gap junction protein expressed in myelinating glial cells. The gene is X‐linked, and the mutations cause a loss of function.AimsA large number of disease‐associated variants have been identified, and many result in mistrafficking and mislocalization of the protein. An existing knockout mouse lacking Gjb1 expression provides a valid animal model of CMT1X, but the complete lack of protein may not fully recapitulate the disease mechanisms caused by aberrant CX32 proteins. To better represent the spectrum of human CMT1X‐associated mutations, we have generated a new Gjb1 knockin mouse model.MethodsCRISPR/Cas9 genome editing was used to produce mice carrying the R15Q mutation in Gjb1. In addition, we identified a second allele with an early frame shift mutation in codon 7 (del2). Mice were analyzed using clinically relevant molecular, histological, neurophysiological, and behavioral assays.ResultsBoth alleles produce protein detectable by immunofluorescence in Schwann cells, with some protein properly localizing to nodes of Ranvier. However, both alleles also result in peripheral neuropathy with thinly myelinated and demyelinated axons, as well as degenerating and regenerating axons, predominantly in distal motor nerves. Nerve conduction velocities were only mildly reduced at later ages and compound muscle action potential amplitudes were not reduced. Levels of neurofilament light chain in plasma were elevated in both alleles. The del2 mice have an onset ~3 months of age, whereas the R15Q mice had a later onset at 5‐6 months of age, suggesting a milder loss of function. Both alleles performed comparably to wild type littermates in accelerating rotarod and grip strength tests of neuromuscular performance.InterpretationWe have generated and characterized two new mouse models of CMT1X that will be useful for future mechanistic and preclinical studies.This article is protected by copyright. All rights reserved.